DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Priority
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Claim Objections
Claims 1, 4, 5, 11, and 14 are objected to because of the following informalities:
Claim 1, L8: “…changing a temperature of each of the micro partitions and measuring…”
Claim 4, L3: “…partitions
Claim 5: “…measuring [[a]] the fluorescence intensity the fluorescence intensity
Claim 11, L4-5 and Claim 14, L4-5: “…which binds to the amplification product of the forward…”
Appropriate correction is required.
Claim Interpretation
PCR amplification is performed by conducting cycles of temperature change during which time primers anneal to a template, are extended, and the product denatured into single stranded form which can be used as template for subsequent cycles. When performing post-PCR melting curve analysis, an additional period of temperature change is conducted in order to measure the change in fluorescence that occurs as a result of a probe melting/denaturing from its target.
Claim 9 is ambiguous regarding if the “cycle of temperature change” is referring to one of the amplification cycles or the additional step of temperature change performing when conducting melting curve analysis. Either will be considered to read on the claim.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 11-15 are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Renard [US 20100280134 A1].
Renard discloses a kit for detecting a predisposition to, or the incidence of, bladder cancer in a sample. This kit contains primer pairs for target DNA, molecular beacon probes, a suitable polymerase for nucleic acid amplification and a stabilizing buffer comprising DMSO [Renard, 0037, 0129-0136].
Renard teaches that molecular beacons contain fluorescent and quenching dyes and are designed to adopt a hairpin structure while free in solution to bring both dyes in close proximity for FRET to occur. When the beacon hybridizes to its target, the dyes are separated and an increase in fluorescence is observed [Renard, 0067].
Regarding claim 14, the method of claim 1 at its core involves amplifying a target using a primer set and performing identification using a fluorescent probe. The method as claimed does not require the primers or probe to be modified in a novel manner. As such, any primer set or probe could be used in the method of claim 1. As the kit of Renard includes the components of claim 14, it too is capable of use in claim 1’s method and therefore anticipates the kit of claim 14.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1-4 and 6-10 are rejected under 35 U.S.C. 103 as being unpatentable over Nakagawa [Nakagawa T et al. Analytical chemistry. 2020 Aug 7;92(17):11705-13] in view of Gene Link [Gene Link. “PCR Additives & Enhancers”. Certificate of Analysis & Product Manual. 2022: p1-12] as evidenced by ThermoFisher [ThermoFisher. “QuantStudio™ 3D Digital PCR Master Mix v2”. Certificate of Analysis. 2017 Jun 9: p1].
Nakagawa teaches a method for performing multiplex genotyping using digital PCR (dPCR) and melting curve analysis. By following this method, Nakagawa was able to simultaneously identify wild-type KRAS, BRAF, and eight mutants of these genes (G12D, G12R, G12V, G13D, G12A, G12C, G12S, and V600E) [Nakagawa, abstract].
First, a sample was partitioned into a chip comprised of 20,000 microwells. Next, asymmetric PCR was then performed within each well. After completion of PCR, target-specific molecular beacon probes were hybridized to the amplicons. As the temperature was decreased from 85oC to 55oC in increments of 2.0oC/min, melting curve analysis was performed by collecting fluorescent images of the chip, reflecting the binding of the probes to their target and allowing for a determination of melting temperature for each probe-target complex. Finally, genotyping was performed using the fluorescence intensity, the fluorescence (dye) color, and the melting temperature [Nakagawa, Fig. 1, p11707].
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Nakagawa teaches the pre-partitioned samples contained target-specific forward and reverse primers, target-specific fluorescent probes, and template DNA obtained from either genomic DNA standards or cfDNA standards [Nakagawa, p11707, Table S2]. The samples also contained 1X QuantStudio™ 3D Digital PCR Master Mix v2 which is composed of DNA polymerase, dNTPs, ROX passive reference, and buffer components [ThermoFisher, CoA].
Nakagawa does not teach including an additive for preventing DNA secondary structure formation in this reaction solution. However, according to Nakagawa, dPCR particularly suffers from efficiency issues and is characterized by large variation in the fluorescence intensity of each partition due to insufficient PCR products. In fact, Nakagawa’s combination of dPCR and melting curve analysis was proposed in an effort to reduce the influence of PCR efficiency on identification capabilities [Nakagawa, p11706].
Gene Link teaches that GC-rich DNA sequences possess high thermal and structural stability which permits secondary structures to form and interferes with the ability of DNA polymerases to accurately amplify those regions. However, this issue can be overcome by adding a PCR additive or enhancing reagent to the reaction mix which increases the yield, specificity, and consistency of PCR reactions. Some examples include 7-deaza dGTP, betaine, dimethyl sulfoxide (DMSO), formamide, and non-ionic detergents (e.g., Triton X-100, Tween 20) which all function to reduce secondary structure formation [Gene Link, p3].
Therefore, a person of ordinary skill in the art prior to the effective filing date of the claimed invention, would have been motivated to include a PCR additive or enhancer in the reaction mix taught by Nakagawa in order to improve PCR efficiency and reduce its overall impact on Nakagawa’s method leading to more accurate and sensitive identification of mutants. A rationale to support a conclusion that a claim would have been obvious is that all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination would have yielded nothing more than predictable results to one of ordinary skill in the art. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395 (2007) (see MPEP §§ 2143, A. and 2143.02).
Regarding claim 2, Nakagawa’s Table S2 discloses the sequence, the fluorescent dye, and the quencher dye used for each probe.
Regarding claim 3, Nakagawa’s Table S2 states that all probes comprise four hydrophobic DNA analogs at each end (i.e., interacting structures). As shown in figure 1, this would bring the ends together when not bound to a target resulting in the quenching of fluorescence due to the proximity of the fluorescent and quenching dyes.
Regarding claim 4, in dPCR, a sample solution containing target DNA is divided into wells before PCR so that each well contains one or zero copies of DNA. If after amplification the well is positive for a given target, then it can be assumed that that well represents one copy of the target. By counting all the positive wells, the skilled artisan is able to determine the copy number of the original sample. By using a combination of the fluorescence intensity, probe dye color, and melting temperature information of each well, Nakagawa was able to determine exactly how many copies of each target were present in the original sample [Nakagawa, p11705-11706].
Regarding claim 9, Nakagawa’s reaction protocol for amplification comprised 60 cycles where each cycle was 95oC for 15 seconds and 60oC for 30 seconds. Additionally, once amplification was complete, the samples were cooled from 85oC to 55oC in increments of 2.0oC/min during which time fluorescence was measured and used to generate melting curves [Nakagawa, p11707].
Regarding claim 10, Nakagawa’s Table S2 indicates the dye used to label each probe and teaches that multiple probes can be labeled with the same fluorescent dye. For example, there are three probes which are labeled with FAM. While Nakagawa developed a prototype imaging instrument capable of capturing fluorescent images in four different channels and thus developed an assay in which probes were labeled with four different dyes, the skilled artisan would recognize that the number of dyes which can be used would be limited by the capabilities of their specific imagine instrument and would adjust the assay accordingly [Nakagawa, p11706].
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Nakagawa and Gene Link (as evidenced by ThermoFisher) as applied to claim 1 above, and further in view of Sinha [Sinha M et al. SLAS TECHNOLOGY: Translating Life Sciences Innovation. 2018 Dec;23(6):580-91].
Nakagawa, Gene Link, and ThermoFisher are applied to the relevant teachings of claim 1 as discussed above and are incorporated herein by reference. Figure 2 of Nakagawa appears to show that imaging of the partitions was performed every 5oC for a total of 7 images. Therefore, Nakagawa does not teach measuring fluorescent intensity at a sampling interval of less than 1oC.
Sinha designed a high-resolution digital melt platform capable of generating up to 20,000 high-resolution melt curves in about 5 minutes. In testing the platform, fluorescent images were captured at an imaging rate that was adjusted based upon the heat ramping rate in order to maintain a resolution of 0.1oC between images (i.e., one image per 0.1 oC). The resulting melt profiles were shown to be highly repeatable across a range of melt rates and commensurate imagining frequencies. Sinha further teaches that increasing the sampling rate for fluorescent data improves a system’s ability to resolve smaller melting temperature differences [581-582, 589]. This is of particular importance for multiplex systems, like that of Nakagawa, which rely on discriminating between melting temperatures in order to accurately genotype a sample. As more targets are added to each dye channel, more sensitive and accurate detection is required. Therefore, a person of ordinary skill in the art prior to the effective filing date of the claimed invention, would have been motivated to perform fluorescence imaging at a sampling interval of 1oC, as taught by Sinha, in order to improve the resolution of Nakagawa’s system resulting in more accurate and sensitive genotyping. The combination of familiar elements is likely to be obvious when it does no more than yield predictable results. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395 – 97 (2007) (see MPEP § 2143, A.).
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1-10 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 4, 5, and 8 of U.S. Patent No. US 12018319 B2 (Tanaka) in view of Nakagawa and Gene Link as evidenced by ThermoFisher.
Both Tanaka and the instant application claim a DNA detection method in which a DNA solution comprising a fluorescent-labeled probe and a target DNA are partitioned into a plurality of compartments in which nucleic acid amplification is performed. By measuring fluorescence intensity in association with a temperature change, a melting temperature of the probe/target complex is calculated.
Tanaka differs from the instant application in that it does not claim that the reaction solution additionally comprises a primer set, an enzyme, and an additive; it does not determine the presence/absence and/or type of target DNA present based on the fluorescence color, fluorescence intensity, and melting temperature; and it additionally calculates a ratio of fluorescence intensity at a first and second temperature.
However, Nakagawa teaches a method for performing multiplex genotyping using digital PCR (dPCR) and melting curve analysis [Nakagawa, abstract]. First, a sample was partitioned into a chip comprised of 20,000 microwells. Next, asymmetric PCR was then performed within each well. After completion of PCR, target-specific molecular beacon probes were hybridized to the amplicons. As the temperature was decreased from 85oC to 55oC in increments of 2.0oC/min, melting curve analysis was performed by collecting fluorescent images of the chip, reflecting the binding of the probes to their target and allowing for a determination of melting temperature for each probe-target complex. Nakagawa also normalized the fluorescent data in order to account for in-plane and well-to-well variation by calculating the ratio of fluorescence intensity at 50oC and at 85oC. Lastly, genotyping was performed using the fluorescence intensity, the fluorescence (dye) color, and the melting temperature [Nakagawa, Fig. 1, p11707, 11709].
Finally, Nakagawa teaches the pre-partitioned samples contained target-specific forward and reverse primers, target-specific fluorescent probes, and template DNA obtained from either genomic DNA standards or cfDNA standards [Nakagawa, p11707, Table S2]. The samples also contained 1X QuantStudio™ 3D Digital PCR Master Mix v2 which is composed of DNA polymerase, dNTPs, ROX passive reference, and buffer components [ThermoFisher, CoA]. While Nakagawa does not teach including an additive for preventing DNA secondary structure formation in this reaction solution, Gene Link teaches that the inclusion of a PCR additive or enhancing reagent to the reaction mix increases the yield, specificity, and consistency of PCR reactions as discussed in the above 35 USC 103 rejection [Gene Link, p3].
Therefore, a person of ordinary skill in the art could have arrived at the claimed invention by modifying Tanaka with the teachings of Nakagawa, Gene Link, and ThermoFisher and would have motivated to do so in order to deleterious effects of PCR inefficiencies and accurately identify and quantify DNA targets.
Claims 1-10 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-12 of copending Application No. 18/582,764 in view of Nakagawa and Gene Link.
Copending 764 describes a method for genetic analysis in which a reaction solution comprising mutation specific probes, a primer, a test biological sample, and an enzyme is used to perform an amplification reaction. This is followed by measuring the binding of an amplicon to a probe via melting curve analysis to determine if the mutations are present in-cis or in-trans. This could be considered the same as determining a type of the target DNA as is required by the instant application. However, copending 764 does not disclose the use of an additive in the reaction mix nor that the reaction mix should be partitioned prior to amplification.
However, as previously described, Nakagawa teaches partitioning samples prior to amplification as part of performing digital PCR which allows for absolute quantification of a target. Gene Link teaches the inclusion of a PCR additive or enhancing reagent to a reaction solution to overcome the effects of PCR inefficiencies. Therefore, a person of ordinary skill in the art could have arrived at the claimed invention by modifying copending 764 with the teaching of Nakagawa and Gene Link and would have been motivated to do so in order to accurately determine the exact quantities of a target within a sample.
This is a provisional nonstatutory double patenting rejection.
Claims 11-15 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 13 of copending Application No. 18/582,764 in view of Gene Link.
Both the instant application and copending 764 claim kits comprised of at least one primer pair and probe. The instant application additionally includes an additive for preventing DNA secondary structure formation. As previously described, Gene Link teaches the inclusion of a PCR additive or enhancing reagent to a reaction solution overcomes the effects of PCR inefficiencies and a skilled artisan would have been motivated to include one in a kit in order to achieve this positive effect in analyses performed with said kit.
This is a provisional nonstatutory double patenting rejection.
Conclusion
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/K.N.K./ Examiner, Art Unit 1681
/SAMUEL C WOOLWINE/ Primary Examiner, Art Unit 1681
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